Bacillus anthracis produces highly lethal disease. Unfortunately, antibiotic treatment options remain few, and therapy must be continued for extended periods. These observations highlight the importance of preserving susceptibility to current antibiotic therapies. However, the very technologies that enable genetic manipulation of B. anthracis for scientific study of its virulence properties rely on introduction of antibiotic resistance. This is in the form of antibiotic resistance markers that permit selection of desired recombination events and maintenance of plasmids. Therefore, our current methods of scientific inquiry might limit therapeutic options, endanger researchers, and even provide ready made resistant pathogens for terrorists. The goals of this proposal are two fold: to develop non-antibiotic based selectable marker systems for use in Bacillus anthracis and to develop genetic systems using these markers that will dramatically increase the efficiency of genetic manipulation of this organism. Although being developed for our ongoing investigation of anthrax pathogenesis, these tools will be broadly applicable to a wide range of other bacterial pathogens. Proposed is one specific aim in three parts. In the first, we will define the ability of markers conferring resistance to bacteriocins, heavy metals, chemotherapeutics, and herbicides to function as selectable markers in Bacillus anthracis. Analysis will include study of the markers'activity and stability, as well as an assessment of potential undesirable properties such as induction of cross resistance to antibiotics and effects on virulence. In the second, we will develop new counterselectable markers that serve as a potent tool for deleting genes, since when introduced into the chromosome they enable selection of rare recombination events in which the marker and linked genes have been deleted. As counterselectable markers do not yet exist for B. anthracis and most other gram positive organisms, we propose development of two innovative counterselectable marker systems. These new markers will greatly accelerate discovery, as they will bring new efficiencies to genetic manipulation of B. anthracis. In the third, we will use newly characterized non-antibiotic selectable markers to create a series of vectors that will serve as new tools for bacterial genetic investigation of Bacillus anthracis and other pathogens. PUBLIC HEALTH RELEVANCE: The goals of this proposal are to develop new tools for analyzing gene function in Bacillus anthracis, the cause of anthrax. The new methods will provide an alternative to the introduction of antibiotic resistance into pathogens, previously needed for this type of research. These new tools will be safer for researchers and the public, accelerate discovery, and find broad use in investigation of a wide range of infectious organisms.